The commercial use of genetically engineered crop species has caused concerns about the possible transfer of transgenes and traits encoded by transgenes from genetically modified plants (GM plants) into landraces, wild relative or other non-GM plant varieties or related crop species (Ellstrand, N.C., 2001, Plant Physiol. 125, 1543-1545; “Quist & Chapela, 2001, Nature, 414, 541-543), which could change the ecological balance in the affected ecosystems or lead to other, first of all, socioeconomic problems. Additionally, there is a certain fear that transgenes, especially antibiotic resistance genes used as transformation markers, can escape, through so-called horizontal transfer, into surrounding microorganisms (Chiter et al. 2000, FEBS Lett., 481, 164-168), thus modifying the microflora in an undesirable way.
Although many of these worries are not well justified scientifically (Christou, P., 2002, Transgenic Res., 11, iii-v), the creation of safe and controlled transgene management systems is highly desirable, as it might prevent potential problems in the future and shall help to protect the germplasm of existing plant species in the most efficient way. In addition, there are problems caused by contamination of organically grown crops or non-GM crops with transgenic cultivars. This has a serious impact on the marketing of transgenic as well as non-transgenic crops, an issue which cannot be ignored by producers.
Unlike other products generated by humans, products created by biotechnology are potentially self-replicating machines. Therefore, any transgenic material created by current technology and released into the environment has a potential of persisting there for a very long time. Common practice of plant genetic engineering is based on the use of expression cassettes and vectors that contain continuous coding sequence for the gene of interest. Such expression cassettes are integrated into a host chromosome and upon hybridization or another genetic information exchange between a GM plant and another organism, whether licit or illicit, the expression cassette is transmitted with a high probability to the progeny or another recipient as a functional transcriptional unit.
WO00/52146 describes general ideas for encrypting a trait of interest by splitting gene(s) in two or more fragments and rejoining the fragments by trans-splicing after mating parental organisms, whereby the parental organisms provide said fragments. WO00/52146 does not go beyond general ideas. It does not contain an enabling disclosure on how these ideas can be reduced to practice. Notably, it does not contain an example. WO00/71701 describes assembly of a functional protein by intein-mediated protein trans-splicing/interaction for improving containment of a transgene encoding said protein. WO00/71701 does not describe bringing together fragments of a protein by mating parent organisms. Further, the frequency of transmission of transgene according to WO00/71701 is not sufficiently low for large scale applications like agriculture, notably when a transgene provides a selective advantage.
WO0116287 relates to the creation of allelic position for transgenes, whose expression determines a phenotype, with the aim that the transgenes segregate to different gametes. This patent application does not address the problem of controlling movement of transgenes, but rather trait generation, specifically male-sterility, encoded by at least two transgenes. Further, it does not mention intein-mediated trans-splicing. Moreover, this application does not describe control over trait movement by splitting a trait-encoding gene in two or more fragments.
Trait assembly from parts encoding the trait is not of high value without knowing how to achieve the most favorable positions of the encoding fragments in practically the most feasible way, in order to provide the strictest control over undesired transmission of said trait. For large scale applications like for agriculture, biological safety requires that undesired transmission of a transgene is reduced to a frequency of practically zero.
Crop plants expressing as a trait of interest male or female sterility are widely used for hybrid seed production. Hybrid crops have on average 20% yield advantage over inbred varieties and production of hybrid seeds is a large industry. Many different technologies are used to produce hybrid seeds (for review see: Perez-Prat E. & van Lookeren Campagne, M M, 2002, Trends Plant Sci., 7 199-202). These technologies can be conditionally divided into at least four groups according to the pollination control mechanism: mechanical, chemical, genetic and transgenic. However, one critical requirement is common for all these technologies: ideally, a 100% male sterile line should be used for the hybridization process and 100% male fertility restoration in F1 progeny should be achieved. Such stringent requirements are absolutely necessary for producing hybrid seeds free of contamination with selfed seeds.
The current methods of hybrid seed production are unsatisfactory in the above respect. These processes are either expensive, as in the case of mechanical de-tasselling (castration) of corn, or “leaky” as in the case of genetic approaches or both as in the case of chemical treatment-based method (e.g. U.S. Pat. No. 4,569,688).
Genetic approaches preferably include the use of lines with cytoplasmic male sterility (CMS) mutants and fertility restorers (e.g. WO02098209). Transgenic approaches use predominantly plants with genetically engineered nuclear male sterility (NMS) or CMS and fertility restoration in F1 progeny (WO8910396; U.S. Pat. Nos. 5,530,191; 6,255,564; WO9832325; WO9201799; U.S. Pat. No. 63,921,191; WO0116287). These approaches also require the use of a so-called maintainer line in order to propagate and maintain the male-sterile line.
The transgenic systems built on one transgene providing for male sterility and another transgene carrying the function of restoring male fertility (e.g. U.S. Pat. No. 62,555,640) guarantee neither complete restoration of male fertility in hybrid progeny nor complete elimination of potentially negative effects of the transgene providing for male sterility on the general health of said progeny. In other words these systems are leaky. In addition, none of the systems mentioned above offers a convenient way of producing and maintaining the male-sterile line. This is an important element of any genetically engineered system for hybrid seed production, as the successful application of such a system for large-scale production depends on whether the male-sterile female parent line can be propagated in an economical and efficient way. In other words, currently there is no universal, reliable and economical system for hybrid seed production, which integrates all requirements necessary for maintenance of the original lines, hybridization process, restoration of male fertility in hybrid progeny and at the same time has high biological safety parameters, e.g. provides for tight control over transgene segregation. A general scheme of hybrid seeds production using currently existing genetic/transgenic approaches is shown in FIG. 12.
In the present invention, we describe a new process of producing hybrid seeds (FIG. 13) which has all necessary characteristics to match the requirements of an ideal hybridization system. A comparison of the hybrid seed production system of the invention with prior art methods is presented in Table 1.
It is therefore an object of the invention to provide a process of producing a transgenic plant expressing a trait of interest, notably male sterility, whereby distribution of said trait to progeny is strictly controlled and occurs with low probability.
It is a further object of the invention to provide a process of producing a biologically safe transgenic plant, notably a male sterile plant, that expresses a trait of interest, whereby gene fragments encoding said trait are positioned such that undesired transmission of said trait occurs with low probability.
It is a further object of the invention to provide a process of positioning transgenic DNA sequences on homologous chromosomes, notably in the same locus of homologous chromosomes of a multi-cellular organism.
It is also an object of the invention to provide a process of producing a male sterile plant line.
It is another object of the invention to provide a universal and environmentally safe process of producing hybrid seeds using a sterile plant line, whereby complete fertility restoration occurs in said hybrid seeds.